Maintenance therapy containing Methotrexate (MTX) and 6-Mercaptopurine (6MP) is essential to cure acute lymphoblastic leukemia (ALL). Cytotoxicity is elicited by incorporation of thioguanine nucleotides (TGN) into DNA (DNA-TG), and higher leucocyte DNA-TG is associated with increased relapse-free survival. As 6-Thioguanine (6TG) provides 6-fold higher cytosol TGN than 6MP, we added low-dose 6TG to MTX/6MP maintenance therapy to explore if this combination results in significantly higher DNA-TG. Target population of the "Thiopurine Enhanced ALL Maintenance therapy" (TEAM) study was n=30 patients, with non-high risk ALL, aged 1-45 years on MTX/6MP maintenance therapy receiving no other systemic chemotherapy. Incremental doses of 6TG were added to MTX/6MP maintenance therapy (start 6TG: 2.5 mg/m2/day, maximum: 12.5 mg/m2/day). Primary endpoint was DNA-TG increments. Thirty-four patients were included, and 30 patients completed maintenance therapy according to TEAM strategy. Of these 30 patients, 26 (87%) tolerated 10.0-12.5 mg/m2/day as maximum 6TG dose. TEAM resulted in significantly higher DNA-TG, when compared with both TEAM patients before TEAM inclusion (on average 251 fmol/μg DNA higher (95% CI 160-341; P<0.0001), and with historical patients receiving standard MTX/6MP maintenance therapy (on average 272 fmol/μg DNA higher (95% CI 147-398; P<0.0001). TEAM did not increase myelotoxicity or hepatotoxicity. Conclusively, TEAM is an innovative and feasible approach to improve maintenance therapy and results in higher DNA-TG without inducing additional toxicity. It may therefore be an effective strategy to reduce risk of ALL relapse through increased DNA-TG, and this will be tested in a randomized ALLTogether-1 substudy.